Physiological Reports ISSN 2051-817X
ORIGINAL RESEARCH
Intratracheal instillation of pravastatin for the treatment of
murine allergic asthma: a lung-targeted approach to deliver
statins
Amir A. Zeki1,2,3,4, Jennifer M. Bratt1,2,3,4, Kevin Y. Chang1, Lisa M. Franzi1,2,3,4, Sean Ott1,2,3,4,
Mark Silveria5, Oliver Fiehn5,6, Jerold A. Last1,2,3,4 & Nicholas J. Kenyon1,2,3,4
1
2
3
4
5
6
University of California, Davis, California
Department of Internal Medicine, University of California, Davis, California
Division of Pulmonary, Critical Care and Sleep Medicine, University of California, Davis, California
Center for Comparative Respiratory Biology and Medicine (CCRBM), University of California, Davis, California
U.C. Davis, West Coast Metabolomics Center (WCMC), University of California, Davis, California
King Abdulaziz University, Biochemistry Department, Jeddah, Saudi Arabia
Keywords
Airway hyperreactivity, airway
hypersensitivity, airway inflammation,
asthma, asthma treatment, goblet cells,
inhaled statin, intratracheal statin, mass
spectrometry, pravastatin, remodeling.
Correspondence
Amir A. Zeki, Genome and Biomedical
Sciences Facility (GBSF), University of
California, 451 Health Sciences Drive,
Room 6517, Davis, CA 95616.
Tel: (530) 754 5469
Fax: (530) 752 8632
E-mail: aazeki@ucdavis.edu
Funding Information
This study was funded by NIH/NHLBI T32 HL07013 (AAZ, JMB), NCATS TR000002
(AAZ), KL2 RR 024144 (AAZ),
1K08HL114882-01A1 (AAZ), HL105573
(NJK), American Thoracic Society Fellows
Career Development Award (AAZ),
Department of Defense W81XWH-10-1-0635
(OF), and LUNGevity Foundation (OF), U24
DK097154 (OF).
Received: 20 February 2015; Accepted: 4
March 2015
Abstract
Systemic treatment with statins mitigates allergic airway inflammation, TH2
cytokine production, epithelial mucus production, and airway hyperreactivity
(AHR) in murine models of asthma. We hypothesized that pravastatin delivered intratracheally would be quantifiable in lung tissues using mass spectrometry, achieve high drug concentrations in the lung with minimal systemic
absorption, and mitigate airway inflammation and structural changes induced
by ovalbumin. Male BALB/c mice were sensitized to ovalbumin (OVA) over
4 weeks, then exposed to 1% OVA aerosol or filtered air (FA) over 2 weeks.
Mice received intratracheal instillations of pravastatin before and after each
OVA exposure (30 mg/kg). Ultra performance liquid chromatography – mass
spectrometry was used to quantify plasma, lung, and bronchoalveolar lavage
fluid (BALF) pravastatin concentration. Pravastatin was quantifiable in mouse
plasma, lung tissue, and BALF (BALF > lung > plasma for OVA and FA
groups). At these concentrations pravastatin inhibited airway goblet cell
hyperplasia/metaplasia, and reduced BALF levels of cytokines TNFa and KC,
but did not reduce BALF total leukocyte or eosinophil cell counts. While pravastatin did not mitigate AHR, it did inhibit airway hypersensitivity (AHS). In
this proof-of-principle study, using novel mass spectrometry methods we
show that pravastatin is quantifiable in tissues, achieves high levels in mouse
lungs with minimal systemic absorption, and mitigates some pathological
features of allergic asthma. Inhaled pravastatin may be beneficial for the
treatment of asthma by having direct airway effects independent of a potent
anti-inflammatory effect. Statins with greater lipophilicity may achieve better
anti-inflammatory effects warranting further research.
doi: 10.14814/phy2.12352
Physiol Rep, 3(5), 2015, e12352,
doi: 10.14814/phy2.12352
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
This is an open access article under the terms of the Creative Commons Attribution License,
which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
2015 | Vol. 3 | Iss. 5 | e12352
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A. A. Zeki et al.
Inhaled Statin for the Treatment of Asthma
Introduction
Asthma affects 27 million Americans, the World Health
Organization estimates that 235 million people have
asthma worldwide, and the prevalence continues to rise.
Investigators are actively exploring new treatments for
asthma, in particular novel inhaler therapies. We and others have previously shown that systemic treatment with statins in animal models of asthma (Zeki et al. 2009) and
chronic obstructive pulmonary disease (COPD) (Davis
et al. 2013) mitigates inflammation and early features of
airway remodeling (Zeki et al. 2010). In this current work,
we explore whether delivering statins directly to the lungs
by intratracheal (i.t.) instillation can mitigate experimental
asthma.
The statin drugs (‘statins’), which inhibit the 3hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR)
enzyme, are highly effective medications for the treatment
of atherosclerosis and cardiovascular diseases. Statins inhibit HMGCR, the rate-limiting step of cholesterol biosynthesis in the mevalonate (MA) cascade. Statins also
exhibit pleiotropic properties that result in a wide range
of cellular and physiological effects by both HMGCRdependent and -independent mechanisms (Wang et al.
2008; Zeki et al. 2011). They have also garnered wide
interest giving rise to studies investigating their therapeutic benefits in different diseases including lung diseases
such as asthma, COPD, pulmonary hypertension, lung
cancer, pneumonia, and bronchiectasis.
In both cell culture experiments and animal models,
systemic administration of the statins (including simvastatin, lovastatin, pravastatin, and atorvastatin) manifests
beneficial anti-inflammatory (Kim et al. 2007; Imamura
et al. 2009), anti-proliferative (Takeda et al. 2006; Capra
and Rovati 2014), anti-fibrotic (Watts et al. 2005), antioxidant (Mctaggart 2003; Shishehbor et al. 2003; Melo
et al. 2013), and immunomodulatory properties (Greenwood et al. 2006; Morimoto et al. 2006). In preclinical
models of asthma, systemic administration of statins (i.e.,
oral and/or intraperitoneal) inhibits allergic inflammation
(Zeki et al. 2009), AHR, goblet cell metaplasia/hyperplasia, and profibrotic changes in airways (McKay et al.
2004; Yeh and Huang 2004; Chiba et al. 2008a; Ou et al.
2008; Ahmad et al. 2011; Huang et al. 2013).
Human observational studies in patients with asthma
indicate a positive correlation between improved lung
function, fewer exacerbations, and reduced corticosteroid
use in statin users versus nonusers (Alexeeff et al. 2007;
Huang et al. 2011; Tse et al. 2013, 2014). However, several small clinical trials in asthma have not confirmed
such statin benefits despite showing reduced sputum
inflammatory cytokine levels and cell counts, and
improvement in quality of life survey scores (Hothersall
2015 | Vol. 3 | Iss. 5 | e12352
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et al. 2008; Fahimi et al. 2009; Cowan et al. 2010; Maneechotesuwan et al. 2010; Braganza et al. 2011; Moini et al.
2012). Despite the limitations of these randomized clinical
trials (RCTs) (e.g., short treatment durations, lack of outcome data such as severe exacerbations) interest remains
in the potential beneficial effect of statins in those with
severe asthma (Zeki et al. 2013).
The statins used in these RCTs were given to study subjects via the oral route which is the only approved route
of statin administration in humans. It remains unknown
whether the statins used (simvastatin, atorvastatin)
reached the airway compartment or whether these statins
target the lung directly or indirectly through peripheral
immune mechanisms. Despite the lung being the target
organ, it has not been determined whether orally ingested
statins penetrate into human lungs or airways. Also, it
remains unclear what the relative effects of statins are in
different anatomic compartments, in particular, the blood,
lung parenchyema, and airways. Therefore, it may be beneficial to give statins via inhalation to directly target the
airway epithelium, a central player in asthma pathogenesis.
The potential advantage of this approach would be the use
of lower drug doses than that used systemically, in order
to achieve greater local potency in the airways while
reducing risk of systemic side effects.
Several in vitro studies using different lung resident cell
types have shown statin benefits, including airway smooth
muscle and airway epithelial cells (Sakoda et al. 2006;
Takeda et al. 2006; Murphy et al. 2008; Schaafsma et al.
2011; Iwata et al. 2012). We have shown that mouse tracheal epithelium may be a viable target for statins (Zeki
et al. 2012), where treatment with simvastatin inhibited
the expression of key IL13-inducible genes important in
helper T-cell type 2 (Th2) inflammation, such as eotaxin1 and the monocyte chemotactic peptides.
Based on this knowledge, the direct instillation of a statin
into mouse trachea in vivo is predicted to inhibit allergeninduced airway inflammation and improve lung function,
in a manner comparable to systemic statin treatment (Kim
et al. 2007; Chiba et al. 2008a; Imamura et al. 2009; Zeki
et al. 2009). Therefore, we hypothesized that intratracheal
instillation of a water-soluble statin, pravastatin, would (1)
produce quantifiable levels of pravastatin in blood plasma,
lung tissue, and bronchoalveolar lavage fluid (BALF) using
mass spectrometry; (2) achieve high drug concentrations
locally in the lung with minimal systemic absorption; and
(3) reduce eosinophilic inflammation, goblet cell hyperplasia, and airway hyperresponsiveness.
In this proof-of-principle study, we report a novel
method for quantifying pravastatin in plasma, homogenized lung tissue, and BALF specimens using ultra performance liquid chromatography – mass spectrometry
(UPLC-MS), and show that i.t. pravastatin reduces aller-
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
A. A. Zeki et al.
Inhaled Statin for the Treatment of Asthma
gen-induced airway goblet cell hyperplasia and airway
hypersensitivity (AHS), without significantly affecting
AHR or airway inflammation. Pravastatin administered
i.t. is well-tolerated and detectible in high concentrations
in OVA-exposed mouse lung tissue as compared to
plasma. This work suggests that statins may be targeted
for delivery to the lung and potentially developed as a
novel class of inhaler therapy for the treatment of human
asthma.
volume of pravastatin solution. Tracheal insertion was
confirmed by palpation of the tracheal rings with the needle tip during insertion (Wegesser and Last 2009). Pravastatin (15 mg/kg dose per instillation) was instilled into
the trachea (i.e., intratracheal (i.t.)) as a single dose
30 min prior to and after each OVA exposure to achieve
target total dose of 30 mg/kg (see below). Animals were
allowed to recover in an upright position until ambulatory before being placed back into their cages.
Materials and Methods
Lung physiology and exhaled nitric oxide
measurements
Animals
Male BALB/c mice, 8 to 10 weeks old and weighing
between 18 to 20 g, were purchased from the Jackson Laboratory (Sacramento, CA), and housed in an AALACaccredited facility equipped with HEPA-filtered laminar
flow cage racks. Mice were maintained on a 12-h light/dark
cycle with food and water given ad lib, and were routinely
screened for health status by serology and histology by veterinary staff of the Animal Resource Services. Animals were
housed no more than four mice per cage, and mice were
allowed to acclimate for 1 week prior to starting experiments. All procedures were performed under an IACUCapproved protocol at the University of California, Davis.
Sensitization and exposure of mice to
ovalbumin and treatment with pravastatin
Mice were sensitized by two intraperitoneal (i.p.) injections
of 10 lg/0.1 mL chicken egg albumin (i.e., ovalbumin
(OVA), grade V, ≥98%; Sigma, St. Louis) with alum adjuvant
on days 1 and 14 of the protocol (Temelkovski et al. 1998).
The mice were divided into four groups; OVA+ i.t. PBS
(n = 16), OVA + i.t. Prav (n = 15), FA + i.t PBS (n = 12),
and FA + i.t. Prav (n = 14). Starting on day 28, mice were
exposed to either an aerosol of 1% OVA dissolved in PBS
(8 mL) for 30 min or filtered air. Aerosols were generated by
a Hudson RCI side-stream nebulizer (Teleflex; Research Triangle Park, NJ) and Passport Compressor (Invacare; Sanford,
FL). Exposure was repeated three times per week for 2 weeks
(for a total of six exposures).
Pravastatin (Cayman Chemical; Ann Arbor, MI) was
dissolved in Dulbecco’s phosphate-buffered saline (PBS)
at a concentration of 9 mg/mL (pH 7.45). Thirty minutes
prior to and after each OVA exposure, mice were briefly
anesthetized using Isoflurane (AttaneTM) administered via
inhalation in an enclosed chamber. Once anesthetized,
they were placed in a supine position, then the tongue
and bottom jaw were gently drawn open. A Hamilton
glass syringe (Model #1705, Hamilton; Reno, NV) with a
blunt-tipped 22-gauge needle was loaded with 50 lL
After the completion of the final OVA aerosol or FA exposure, mice were deeply anesthetized with medetomidine
(Domitor 0.75 mg/kg, Orion Pharma; Finland) and tiletamine/zolpidem (Telazol 37.5 mg/kg; Fort Dodge Laboratories; Fort Dodge, IA). Mice were cannulated and ventilated
at 8 mL/kg using a mouse ventilator (MiniVent, Harvard
Apparatus; Cambridge, MA) in a whole body plethysmograph for restrained animals (Buxco, Inc.; Troy, NY). A 5min sample of exhaled breath was collected in Mylar bags
via the exhalation port of the ventilator during baseline
lung physiology measurements, and the fraction of exhaled
nitric oxide (FeNO) was measured in parts per billion
(ppb) by a chemiluminescence assay using the Sievers
Nitric Oxide Analyzer (GE Instruments, Sievers; Boulder,
CO). Placement of the Mylar bag did not affect pressure
measurements.
Lung physiology (dynamic compliance (Cdyn) and respiratory system resistance (Rrs)) was measured by plethysmograph using the BioSystem XA software (Buxco, Inc., Troy,
NY). These parameters were measured in 10 sec increments
and averaged over a 5-min baseline period, a 3-min saline
aerosol exposure period, and a series of 3-min methacholine (MCh) aerosol dose challenges (0, 0.5, 1.0, and 2.0 mg/
mL) (Kenyon et al. 2003) to determine airway hyperreactivity (AHR) and airway hypersensitivity (AHS).
AHR is defined as greater airway reactivity to a given
dose of MCh or over increasing doses of MCh represented by a steeper slope of the dose–response curve; and
AHS is defined as increased airway resistance above baseline occurring at a lower effective dose of MCh than its
respective control (Affonce and Lutchen 2006), that is,
the airways require less contractile stimulus when compared with healthy airways. We use the term “airway hyperresponsiveness” to include both AHR and AHS
(O’Byrne and Inman 2003; Turi et al. 2011).
Blood collection
Mice were killed with an overdose of Beuthanasia-D
(pentobarbital sodium and phenytoin sodium) by
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
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A. A. Zeki et al.
Inhaled Statin for the Treatment of Asthma
intraperitoneal (i.p.) injection at the conclusion of lung
physiology measurements. Blood was collected via cardiac
puncture in heparinized tubes (~1 mL). Plasma was
obtained by centrifugation at 4°C and stored at 80°C
for further measurement of pravastatin by UPLC-MS.
Bronchoalveolar lavage fluid (BALF)
collection and inflammatory cells counts
Mice tracheas were cannulated; and lungs were lavaged
using two 1 mL aliquots of sterile PBS (pH 7.4) containing a general protease inhibitor cocktail with aprotinin
and leupeptin (1:100; Sigma; St. Louis, MO) and
0.1 lmol/L phenylmethanesulfonylfluoride (PMSF), with
each aliquot passaged twice. The collected BALF was centrifuged at 2000 rpm on a bench top centrifuge and the
resulting supernatant was decanted and stored at 80°C
for cytokine analysis and UPLC-MS measurement of pravastatin. The remaining cell pellet was resuspended in
ACK lysis buffer (0.15 mol/L NH4Cl, 1 mmol/L KHCO3,
0.1 mmol/L EDTA, pH 7.3), then centrifuged for an additional 10 min. The subsequent cell pellet was re-suspended in 0.5 mL PBS, and an aliquot of this fluid was
removed for live cell counting.
A 20 lL volume of the cell suspension was used to
determine the BALF total live cell counts using a hemacytometer and Trypan Blue exclusion method. Then a
100 lL volume of the cell suspension was also processed
onto slides using a cytofuge. Slides were air-dried then
stained with a Hema3 stain set per the manufacturer’s
instructions (Fisher Scientific; Kalamazoo, MI). Cell percent differentials were calculated by counting 10 fields at
4009 and classifying cell types as macrophage, neutrophil,
eosinophil, lymphocyte, or “other” based upon standard
morphological characteristics and staining profile.
Bronchoalveolar lavage cytokine levels
The concentrations of selected helper T-cell type 1 (Th1),
Th2, and Th17 cytokines and chemokines from BALF
supernatant were measured with commercially available
multiplex assays (EMD Millipore; Billerica, MA). For
cytokine/chemokine sample measurements below the
lower detection limit, results were assigned a value equal
to the minimal detection limit for the specific assay to
facilitate statistical analysis of the data.
Lung histopathology
After completion of lung lavage, the chest cavity was
exposed and the right bronchus was ligated using surgical
suture. The right lung lobes were isolated and snap frozen
on dry ice for UPLC-MS measurements. The remaining
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intact left lung lobe was fixed for histological examination
in situ under constant hydrostatic pressure of 30 cm H2O
using 1% paraformaldehyde (PFA) in PBS (pH 7.4), and
embedded in paraffin blocks for sectioning.
Lung histolopathology was examined for the qualitative
assessment of peribronchial inflammation and goblet cell
metaplasia/hyperplasia as previously described Zeki et al.
(2010). Paraffin-embedded left lung lobes were sectioned
at a thickness of 5 microns parallel to the main conducting airways. Sections were stained with either Alcian
Blue-Periodic acid Schiff (PAS) or with hematoxylin and
eosin (H&E) to assess the degree of goblet cell hyperplasia/metaplasia or peribronchial and perivascular inflammation, respectively.
Each animal was represented by a single section of lung
that included the 2nd, 3rd, and 4th generations of conducting airways. Five randomly selected regions were evaluated, including 2 segments of the 2nd generation, 2
segments from the 3rd generation, and 1 segment from a
4th generation of conducting airways. A minimum of 100
sequential airway epithelial cells were counted from each
region, and the ratio of PAS positive cells per total epithelial cells was determined for each region. These regional values then were averaged to give a final PAS score
per animal reported as “% Positive PAS Cells”.
Mass spectrometry to measure pravastatin
Blood plasma, BALF, and lung samples were used to
determine pravastatin tissue absorption. Blood and BALF
specimens were processed as described above. Right lung
samples were homogenized on ice using a mixture of
stainless steel and silica beads in a 2010 Geno/Grinder
(Spex SamplePrep; Metuchen, NJ) at 1500 strokes/min.
As part of our method development, the optimal mixture was determined to be an acetonitrile-H2O solution
(1:1, v/v), as compared to isopropanol/acetonitrile/water
or methanol/water. This acetonitrile solution was used to
extract pravastatin from specimens of plasma, BALF, and
whole lung homogenates. Samples were vortexed and centrifuged, the supernatant was transferred to a new tube
and dried completely using a Labconco Centrivap Speedvac, then resuspended in 100 lL of acetonitrile-H2O solution (1:1, v/v). Sample supernatants were analyzed by
ultra performance liquid chromatography – mass spectrometry (UPLC-MS). Separation was performed by a
Waters Acuity UPLC on a Waters HSS T3 reversed phase
100 A, 1.8 lm, 2.1 mm 9 30 mm column. A 6 min gradient (at 0.4 mL/min) of 10 mmol/L ammonium formate
with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) was formed by starting with 30% B, increasing to 95% B at 4 min, followed by 2 min of
equilibration.
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
A. A. Zeki et al.
An AB Sciex Qtrap 4000 mass spectrometer was set to
monitor the most optimum transition for Pravastatin precursor-to-product of m/z 423.2 ([M-H]-) ? m/z 100.8
using MRM (Multiple Reaction Monitoring) in negative
mode. Quantitation was performed for this transition
using a standard curve made from known stock solutions.
Optimized conditions for best sensitivity were found at a
collision energy of 47.7 volts and declustering potential
of 109 volts at its retention time. The limit of detection
(LOD) was determined to be 350 pg/lL.
Inhaled Statin for the Treatment of Asthma
with Tukey’s posttest correction for 1-way ANOVA and
Bonferroni’s posttest correction for 2-way ANOVA. For
nonparametric data, we used the Mann–Whitney or Kruskal–Walis test with Dunn’s posttest correction to determine statistical significance. For unequal variance, Welch’s
correction for t tests was used where appropriate. The Wilcoxon signed rank test was used to analyze median values
with a nonparametric distribution. Data are plotted as
means SEM except where indicated.
Results
Calculating pravastatin concentration in
lung tissue
While pravastatin concentrations in nanogram per gram
of lung (ng/g) would enable us to calculate absolute values (i.e., exact drug concentrations) for pravastatin in
lung tissue, estimating that homogenized mouse lung has
a density similar to (or slightly higher than) water at
≥1 g/mL, units of ng/lL allowed us to compare drug levels between plasma, BALF, and lung tissue samples. We
recognize the limitations of assuming a value of 1 g/mL
density for lung tissue. Variations in density may exist
between OVA and FA groups due to factors such as
edema, inflammation, and mucin production which cannot be addressed in this calculation without previously
determining the lung dry weight, % solubility of the tissue, and final vol/weight ratio of the lung homogenate
(Fig. 1C). However, it is important to note that while the
absolute pravastatin levels (i.e., exact drug concentrations)
would be different, the relative changes in pravastatin partitioning among the different tissue compartments will
likely be the same (or similar) to our results using this
estimate of homogenized lung density.
Statistical analysis
Our results represent data from three independent experiments. Data were analyzed using the Prism 5 software
package (GraphPad, Inc.; San Diego, CA). All data were
tested for normality using the D’Agostino and Pearson
omnibus test. Where appropriate data were log or natural
log (Ln) transformed and re-assessed for normality, then
tested for statistical significance using parametric or nonparametric tests. Where appropriate, data that were two
standard deviations (SDs) outside the mean were considered extreme outliers and excluded from analysis. Mice
with BALF eosinophil counts < 30% in our OVA model are
considered allergen nonresponders; one animal considered
a nonresponder was excluded from analysis. Responders to
OVA in our model typically have at least ≥60% BALF
eosinophilia and on average we observe 70 to 80% eosinophilia. For parametric data, we used the t test or ANOVA
Pravastatin concentration in plasma and
lung tissues
The first question we addressed was whether pravastatin
was measurable in the lung in greater quantities than the
systemic circulation following i.t. instillation. To determine this we used UPLC-MS to quantify pravastatin levels in plasma, BALF, and lung tissue homogenates. The
detection of pravastatin was most sensitive in the negative
ionization mode due to reduced noise levels by coeluting
compounds or matrix components. Pravastatin was readily detected by tandem MS/MS fragmentation of the deprotonated pravastatin precursor ion m/z 423.2 with two
major fragmentation products at m/z 58.8 and m/z 100.8
(acetate and 2-methylbutanoate substructure fragments,
respectively). An example of the MS/MS spectrum of pravastatin is shown in Fig. 1D, using pure drug as a standard. Mice administered the vehicle (PBS) alone did not
have detectible levels of pravastatin in any of the tissue
compartments analyzed, as expected.
Combining the OVA and FA groups and comparing by
tissue compartment, measured levels of BALF pravastatin
were significantly higher than plasma pravastatin. On
average, BALF pravastatin concentration was 35.7-fold
higher than plasma (plasma 0.38 0.11 vs. BALF
13.58 5.21 ng/lL, *P = 0.0039 by Kruskal–Wallis test).
There was no significant difference between the lung
compartment pravastatin concentration (6.87 2.70 ng/
lL) and plasma or BALF pravastatin concentration
(Fig. 1A).
Comparing the OVA vs. FA groups, OVA-exposed mice
exhibited the highest mean concentrations of pravastatin
in the BALF (18.12 9.31 ng/lL) and lung tissue
(10.00 4.96 ng/lL) with minimal pravastatin detected
in plasma (0.26 0.13 ng/lL) indicating low systemic
absorption (Fig. 1B and C). In the OVA groups, mean
pravastatin concentration in BALF is 69.7-fold higher
than plasma (OVABALF 18.12 9.31 vs. OVAplasma
0.26 0.13 ng/lL, **P = 0.0094 by 1-way ANOVA), and
in lung it is nearly 38.5-fold higher than plasma. FAexposed mice exhibited similar trends in tissue distribu-
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
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A. A. Zeki et al.
Inhaled Statin for the Treatment of Asthma
B
(n = 6) (n = 7) (n = 5)
(n = 6) (n = 7) (n = 5)
A
(n = 12)
(n = 10)
(n = 14)
D
C
The animal number ‘n’ is the same as in Panel B.
Figure 1. Pravastatin Quantification Using Mass Spectrometry and Determination of Concentrations in Three Tissue Compartments – Plasma,
BALF, and Lung. (A) Plasma, BALF, and lung pravastatin concentrations were measured combining mice from all four treatment groups (OVA
and FA), plotted by tissue compartment. Pravastatin was not detected in PBS drug vehicle controls. Pravastatin concentration in BALF was 35.7fold higher than plasma (Plasma 0.38 0.11 vs. BALF 13.58 5.21 ng/lL, *P = 0.0039; and for Lung 6.87 2.70 ng/lL vs. Plasma, or Lung
vs. BALF, P = NS by Kruskal–Wallis test). (B) In OVA-exposed mice, whereas the mean pravastatin concentration in BALF was 69.7-fold higher
than plasma, and in lung was 38.5-fold higher than plasma, only OVABALF and OVAplasma showed a statistically significant difference in
pravastatin concentration (**P = 0.0094 by 1-way ANOVA). All other relevant comparisons were not statistically significant (P = NS) (Including
OVABALF vs. FABALF, OVAlung vs. FAlung and OVAplasma vs. FAplasma). (C) This panel is the tabular representation of the graph in panel (B). (D) We
determined the MS/MS spectrum of pravastatin in both lung tissue and plasma using ultra performance liquid chromatography – mass
spectrometry (UPLC-MS), which detected molecular ions in negative ionization mode. This figure shows the spectrum of pravastatin using pure
drug standard, where the molecular structure of pravastatin is indicated at the pravastatin peak (right-side black arrow). Two other
fragmentation products are indicated with black arrows (left side of figure). Abberviations: m/z = mass to charge ratio, Da = Daltons,
cps = counts per second. The number of mice (n) is indicated in parentheses for each experimental group.
tion to their OVA-exposed counterparts, but the differences were not statistically significant. All other relevant
comparisons including compartmental comparisons
between FA- and OVA-exposed groups were not statistically significant, that is, OVABALF vs. FABALF, OVAlung vs.
FAlung, and OVAplasma vs. FAplasma (P = NS by 1-way
ANOVA, Fig. 1B). These results indicate that while the
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majority of pravastatin remains in the lung, there is some
low-level systemic absorption of the drug.
Because of the limitations of calculating pravastatin
concentrations in lung tissue (Fig. 1) based on estimates
of lung density (see Materials and Methods), we also
determined the absolute levels of pravastatin (i.e., exact
drug concentration) in nanograms per gram of lung tissue
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
A. A. Zeki et al.
Inhaled Statin for the Treatment of Asthma
and in nanograms per right lung. While pravastatin levels
were apparently higher in the OVA + i.t. pravastatin
group as compared to the FA + i.t. pravastatin group, this
difference did not reach statistical significance
(14,001 6136 vs. 5221 2439 ng/g; P = NS by Mann–
Whitney test). There was no detectible pravastatin in the
OVA and FA PBS controls, as expected.
Plotted as ng/right lung, OVA+i.t. pravastatin had
higher levels than FA+i.t. pravastatin, 2637 1823 vs.
1641 858.9, respectively, but this did not reach statistical significance (P = NS by Mann–Whitney test). The
amount of pravastatin in the right lung is less than the
ng/g calculation because the right lungs on average
weighed 0.243 0.034 g in this experiment.
Normalized pravastatin levels in OVA- vs.
FA-exposed lungs
To compare relative pravastatin levels in BALF, lung, and
plasma between the FA- and OVA-exposed groups, we
first corrected for the relative differences in plasma pravastatin levels by normalizing BALF and lung tissue pravastatin levels to their respective plasma drug
concentration for each mouse; we then plotted the means
of these normalized values (Fig. 2). For plasma comparisons, normalization was not necessary given it was in one
tissue compartment. This allowed us to account for
potential differences in relative pravastatin absorption
from mouse to mouse, thereby allowing comparisons as
ratios or normalized values for lung and BALF pravastatin
levels.
Normalized BALF pravastatin values were significantly
higher in OVA-exposed mice than FA controls (55.7 vs.
19.5, *P = 0.0158 by t test). While there was a similar
trend in homogenized lung tissue (69.1 vs. 14.6, P = NS
by t test, Fig. 2A), these and other relevant comparisons
between normalized values were not statistically significant (P = NS by ANOVA or t test).
We also compared fold changes in pravastatin levels
in plasma-normalized lung homogenate and BALF
A
(3)
(3)
(3)
(2)
B
Figure 2. The Effect of Allergic Inflammation on Relative Pravastatin Drug Levels in the Same Tissue Compartments. (A) Pravastatin
concentrations for BALF and lung samples were normalized to their respective plasma concentrations for each mouse. In OVA-exposed mice,
pravastatin levels in BALF were significantly higher than FA controls (55.7 vs. 19.5, *P = 0.0158 by t test), with a similar trend in homogenized
lung tissue (69.1 vs. 14.6, P = NS by t test). All other relevant comparisons were not statistically significant (P = NS by ANOVA or t test). (B)
OVA groups are compared to the FA groups across each tissue compartment to determine fold change in mean pravastatin levels. Plasmanormalized BALF pravastatin concentration for the OVA group (i.e., OVABALF) was on average 2.86-fold higher than its respective FABALF group.
Plasma-normalized lung pravastatin concentration for the OVAlung group was 4.73-fold higher than its respective FAlung group. The numbers in
parentheses listed on each bar graph indicate the number (n) of mice per group.
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
2015 | Vol. 3 | Iss. 5 | e12352
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A. A. Zeki et al.
Inhaled Statin for the Treatment of Asthma
between the FA- and OVA-exposed treatment groups.
Fold changes in drug levels due to OVA-induced inflammation gives some indication of the magnitude of pravastatin partitioning when comparing BALF versus lung
tissues. The normalized BALF concentration of pravastatin in the OVA-exposed group (OVABALF) was 2.86-fold
higher than its respective FA group (FABALF) (Fig. 2B).
Normalized lung pravastatin concentration in the OVAexposed group (OVAlung) was 4.73-fold higher than its
respective FA group (FAlung) (Fig. 2B). Lung and BALF
consistently had the highest relative concentration of
pravastatin as compared to plasma, for both OVA and
FA groups.
Pravastatin reduces goblet cell hyperplasia
and metaplasia
After establishing that pravastatin was measurable in the
lung, we assessed its anti-inflammatory potential and
effects on components of airway remodeling. We predicted that the direct application of pravastatin to the airways would attenuate OVA-induced goblet cell metaplasia
and hyperplasia. In OVA-exposed mice, PAS staining
showed a significant reduction in goblet cell hyperplasia
by 30.5% (*P < 0.05, 1-way ANOVA) calculated as “%
Positive PAS Cells” in mice treated with i.t. pravastatin
compared to mice treated with i.t. PBS. There was no statistically significant change in FA control mice with pravastatin treatment (Fig. 3).
Pravastatin improves airway
hypersensitivity but not airway
hyperreactivity
Our hypothesis predicted that instilling pravastatin
directly into airways would result in potent improvements
in airway physiology, that is, inhibition of airway hyperreactivity (AHR) and airway hypersensitivity (AHS), while
preserving lung compliance.
We found that pravastatin had differential effects on
lung mechanics. While pravastatin inhibited AHS, overall
it had no statistically significant effect on AHR. Ovalbumin aerosol exposure increased Rrs and induced AHR as
compared to FA controls (OVA + i.t. PBS vs. FA + i.t.
PBS and vs. FA + i.t. pravastatin; for all three doses of
MCh, P < 0.0001 by 2-way ANOVA). This indicates an
appropriate response to allergen in our model with
respect to airway hyperresponsiveness. However, pravastatin had no statistically significant effect on MCh-induced
AHR except at the 0.5 mg/mL MCh dose (*P < 0.05, by
2-way ANOVA) (Fig. 4A). An independent analysis using
linear regression to assess change in slope of the Rrs data
showed no statistically significant differences with pravast-
2015 | Vol. 3 | Iss. 5 | e12352
Page 8
atin treatment in the OVA groups (P = NS, data not
shown).
Analyzing total Rrs data by treatment group, the OVA
+ i.t. PBS group had the highest airway resistance (or Rrs)
compared to FA + i.t. PBS controls (**P < 0.0001, by
Kruskal–Wallis test). Administration of i.t. pravastatin
reduced the OVA-induced increase in total Rrs by 14.3%
(*P < 0.05, by Kruskal–Wallis) (Fig. 4B). Of note, the
horizontal dotted line in Fig. 4B represents the expected
Rrs for FA controls in our mouse model; it represents a
historical average of data from multiple prior experiments
using our model system and provides a reference point
for the expected Rrs in FA mice.
To measure pravastatin-dependent changes in AHS, we
used the provocative concentrations (PC) of MCh (0.5,
1.0, and 2.0 mg/mL) to cause a 5, 10, and 20% increase
from baseline Rrs (PC5, PC10, and PC20, respectively),
representing low, medium, and high MCh doses in our
model system (Fig. 4C). We used this percentage range
because of the constraints of our plethysmograph system
in terms of the achievable Rrs using only three doses of
MCh: 0.5, 1.0, and 2.0 mg/mL. For the OVA groups, saline challenge (0 mg/mL MCh) followed by these three
doses of MCh increased average Rrs above baseline values
by a maximum of 18.5 to 22% at the 2.0 mg/mL dose.
For the FA groups, this increase ranged from 5.4 to 8.3%
at the 2.0 mg/mL MCh dose. We determined that PC5,
PC10, and PC20 was an appropriate range of % increase
in Rrs fitting our model system, where PC10 is approximately half of the increase in Rrs values above baseline.
Therefore, within the constraints of our model, PC10 and
the range we chose (PC5 to PC20) is a meaningful measure.
The MCh provocative concentrations were higher for
pravastatin- than PBS-treated OVA mice for PC10
(*P = 0.04) and PC20 (**P = 0.024 by Wilcoxon
signed rank test). For PC10 and PC20 challenges, it
takes twice the dose of MCh to induce an equivalent %
increase in Rrs in the pravastatin-treated mice as compared to PBS controls. Therefore, treatment with i.t.
pravastatin significantly decreased AHS at the medium
and high PC doses in OVA-exposed mice. There were
no significant differences in the FA groups (P = NS,
Fig. 4C).
In our mouse model, OVA exposure decreases Cdyn
due to increased airway inflammation, edema, and
mucus production. As expected, OVA sensitization and
exposure decreased Cdyn relative to FA controls (OVA +
i.t. PBS vs. FA + i.t. PBS, P < 0.001 for MCh doses 0.5
and 1 mg/mL, and P < 0.0001 for 2 mg/mL, by 2-way
ANOVA. For OVA + i.t. PBS vs. FA + i.t. pravastatin,
P < 0.05 for MCh doses 0.5 and 1.0 mg/mL, and
P < 0.01 for dose 2.0 mg/mL, by 2-way ANOVA). While
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the American Physiological Society and The Physiological Society.
A. A. Zeki et al.
Inhaled Statin for the Treatment of Asthma
A
B
(5)
(4)
(4)
(5)
Figure 3. Pravastatin Effect on Goblet Cell Hyperplasia/Metaplasia. Treatment with i.t. pravastatin reduced goblet cell hyperplasia/metaplasia
(black arrows, 2009 magnification) by 30.5% (*P < 0.05, 1-way ANOVA) in the OVA group. Pravastatin had no significant effect in the FA
controls (1009 magnification). The numbers in parentheses listed on or above each bar graph indicate the number (n) of mice per group.
this indicates an appropriate response to allergen exposure in our model with respect to airway physiology,
pravastatin had no statistically significant effect on
MCh-induced reductions in Cdyn (P = NS by 2-way
ANOVA) (Fig. 4D).
Pravastatin has selective effects on the antiinflammatory response
We and others have previously shown that giving simvastatin (Zeki et al. 2009) or pravastatin (Imamura et al.
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
2015 | Vol. 3 | Iss. 5 | e12352
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A. A. Zeki et al.
Inhaled Statin for the Treatment of Asthma
A
(n = 7)
C
(n = 9)
(n = 7)
(n = 7)
(n = 8)
(n = 9)
(n = 7)
(n = 8)
D
B
(n = 7)
(n = 9)
(7)
(9)
(7)
(n = 7)
(8)
(n = 11)
Figure 4. The Effect of Intratracheal Pravastatin on Lung Physiology. (A) Pravastatin had no statistically significant effect on methacholine
(MCh)-induced airway hyperreactivity (AHR) except at the 0.5 mg/mL MCh dose (*P < 0.05, by 2-way ANOVA). An independent analysis using
linear regression also showed no statistically significant differences (data not shown). (B) Analyzing averaged respiratory system resistance (Rrs)
data by treatment group, the OVA + i.t. PBS group had the highest airway resistance (or Rrs) compared to FA + i.t. PBS and FA + i.t. Pravastatin
(**P < 0.0001, by Kruskal–Wallis test). Pravastatin reduced the OVA-induced increase in total Rrs by 14.3% (*P < 0.05, by Kruskal–Wallis test).
The horizontal dotted line represents the expected Rrs for FA controls in our mouse model (see Results section for details). (C) The provocative
concentrations (PC) of MCh were higher for OVA + i.t. Pravastatin than OVA + i.t. PBS mice for PC5 (P = NS), PC10 (*P = 0.04), and PC20
(**P = 0.024 by Wilcoxon signed rank test). This is consistent with a decrease in airway hypersensitivity (AHS) due to treatment with
pravastatin. There were no significant differences in the FA groups. Values for the median MCh dose are listed under the PC5 to PC20
columns. (D) Pravastatin did not improve dynamic lung compliance (Cdyn) (P = NS by 2-way ANOVA). The number of mice (n) is indicated in
parentheses for each treatment group.
2009) via the intraperitoneal (i.p.) route significantly
attenuates systemic and airway Th2 allergic inflammation
in OVA models of asthma. We hypothesized that i.t. pravastatin would be more potent at inhibiting Th2 allergic
inflammation given the lung-targeted approach. We tested
this prediction by examining lung histopathology, BALF
total and differential cell counts, BALF cytokine levels,
and exhaled NO levels.
2015 | Vol. 3 | Iss. 5 | e12352
Page 10
Although pravastatin decreased levels of TNFa and KC
in BALF, it did not reduce inflammation in BALF (Fig. 5).
In the OVA groups, pravastatin decreased TNFa by 60.4%
(*P < 0.0001 by 1-way ANOVA) and KC by 48.6%
(*P < 0.0001 by 1-way ANOVA) in BALF (Fig. 5A and B).
Pravastatin treatment had no statistically significant effects
on other BALF cytokines/chemokines including IL-13, IL4, eotaxin, RANTES, IL-5, IL-10, IP-10, IFNc, IL-1a, IL-1b,
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
A. A. Zeki et al.
A
B
(n = 9)
(n = 10)
Inhaled Statin for the Treatment of Asthma
(n = 8)
(n = 8)
(n = 8)
(n = 8)
(n = 10)
D
(n = 10)
C
Figure 5. Pravastatin Effect on Lung and Airway Inflammation. (A) Treatment with pravastatin i.t. reduced BALF concentrations of TNFa by
60.4% (*P < 0.0001 by 1-way ANOVA) in the OVA groups. (B) Pravastatin reduced BALF concentrations of KC by 48.6% (*P < 0.0001 by
1-way ANOVA) in the OVA groups. (C) Pravastatin i.t. had no statistically significant effect on airway leukocyte influx as measured by BALF
total leukocyte (P = NS by Kruskal–Wallis test) or differential cell counts (including BALF absolute eosinophil, lymphocyte, macrophage, and
neutrophil counts (See Table 1)). (D) By qualitative histological assessment on H&E staining, i.t. pravastatin attenuated OVA-induced
peribronchiolar inflammation in OVA-exposed mice (2009 magnification). There were no visible differences between the FA groups (1009
magnification). The number of mice (n) is indicated in parentheses for each treatment group.
or IL-17. Of note, MCP-1 was not detectible in any
treatment group (by ANOVA or t test, data not shown).
Qualitative histological assessment of H&E stained lung
sections of pravastatin-treated mice exhibited a reduction
in OVA-induced peribronchiolar and lung parenchymal
inflammatory cell influx. There were no visible differences
in the FA groups with or without pravastatin treatment
(Fig. 5D). However, pravastatin had no statistically significant effect on the influx of airway leukocytes as measured by BALF total leukocyte counts (P = NS by
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
2015 | Vol. 3 | Iss. 5 | e12352
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A. A. Zeki et al.
Inhaled Statin for the Treatment of Asthma
Kruskal–Wallis test) (Fig. 5C), or inflammatory differential cell counts (Table 1).
For all inflammatory cell types (i.e., eosinophil, macrophage, lymphocyte) except for absolute neutrophil count,
there was a statistically significant difference in the OVA
+ i.t. PBS vs. FA + i.t. PBS groups indicating an appropriate allergic inflammatory response in our model
(P < 0.001, P < 0.05, P < 0.05, and P = NS, respectively).
Intratracheal pravastatin had no statistically significant
effects in both OVA and FA treatment groups for absolute eosinophil, lymphocyte, and neutrophil counts
(P = NS by Kruskal–Wallis test), and for absolute macrophage count (P = NS by 1-way ANOVA; Table 1).
Treatment with pravastatin did not affect FeNO levels
among all four treatment groups (OVA + i.t.
PBS = 4.3 1.6, OVA + i.t. Pravastatin = 4.3 0.8, FA +
i.t. PBS = 3.5 0.5, FA + i.t. Pravastatin = 3.6 0.6 ppb;
P = NS by 1-way ANOVA). Log transformation of the data
and nonparametric analyses also yielded nonsignificant differences (P = NS; data not shown).
Tolerability of intratracheal pravastatin in
mice
Intratracheally instilled pravastatin did not appear to
induce additional damage to the airway epithelial lining
as observed by histological assessment at 4009 magnification (Fig. 6A). There was no histological evidence of
bronchial epithelial denudation or sloughing. Pravastatin
in both FA controls and OVA-exposed mice was well-tolerated as measured by steady weight gain in all mice
(Fig. 6B). There were no statistically significant differences
in baseline weights (Day #1) between treatment groups,
and compared to their own respective baseline values, all
mice in each group gained weight steadily over a period
of 1 to 13 days during drug treatment (Fig. 6B).
Discussion
This is the first study that investigates whether administration of pravastatin directly into the lungs mitigates
experimental allergic airway inflammation and airway hyperresponsiveness, and whether it is quantifiable in
plasma and lung tissues. In this proof-of-principle study,
we show that intratracheally instilled pravastatin achieves
high concentrations in the lung with low systemic distribution, may have therapeutic potential in asthma, and
does not demonstrate acute toxicity in mice. We also
developed a novel mass spectrometry method to measure
pravastatin in lung tissues allowing us to estimate relative
drug distribution. Pravastatin has demonstrated benefits
in our model; however, other statins may be more potent
via the intratracheal (i.t.) route. Additional research is
needed to determine the ideal type of statin and dose to
use which can lead to the development of a novel class of
inhaler therapy for human asthma.
In our prior work, we established that systemic treatment with simvastatin attenuates allergic inflammation in
a MA-dependent manner, decreases AHR, and reduces
hallmarks of adverse airway remodeling in animal models
of asthma (Zeki et al. 2009, 2010). We also showed that
simvastatin directly inhibits IL13-induced expression of
proinflammatory cytokines and chemokines including eotaxin, in primary mouse tracheal epithelial cells (Zeki
et al. 2012). Using murine models, others have shown that
systemic treatment with pravastatin (Imamura et al. 2009)
yields similar antiinflammatory effects to simvastatin
(Joyce et al. 2001; Yeh and Huang 2004; Krauth et al.
2006; Imamura et al. 2009). We considered whether an
airway-targeted approach using pravastatin might also
have anti-inflammatory effects while measuring drug levels
in different tissue compartments to assess its distribution.
For our experiments we selected pravastatin for two reasons: (1) pravastatin’s beneficial anti-inflammatory effect
in the ovalbumin mouse model, when given systemically,
was shown to be similar to simvastatin; and (2) pravastatin
is water soluble, unlike the lipophilic statin simvastatin,
and can easily be dissolved in saline for intratracheal instillation, thereby avoiding the use of drug vehicles that could
irritate or damage airway mucosa.
We thought that targeting the lung via intratracheal
instillation would lower the effective statin doses to
Table 1. BALF leukocyte differential cell counts.
OVA + i.t. PBS
(n = 14–16)
Abs.
Abs.
Abs.
Abs.
Eos. Count
Mac. Count
Lymph. Count
Neutr. Count
2.9
1.5
1.7
2.8
0.54 9 105
0.25 9 105
0.73 9 104
1.1 9 104
OVA + i.t. Pravastatin
(n = 14–15)
1.9
1.3
2.0
3.7
0.44 9 105
0.22 9 105
0.71 9 104
1.6 9 104
FA + i.t. PBS
(n = 12)
92.9
0.59
338.8
0.34
83.4
0.16 9 105
237.6
0.11 9 104
FA + i.t. Pravastatin
(n = 12–13)
730.8
0.49
286.5
0.15
337.9
0.07 9 105
208.3
0.07 9 104
P = NS for all OVA + i.t. PBS vs. OVA + i.t. Pravastatin, and similarly for FA groups.
Abs. (absolute), Eos. (eosinophil), Mac. (macrophage), Lymph. (lymphocyte), and Neutr. (neutrophil).
2015 | Vol. 3 | Iss. 5 | e12352
Page 12
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the American Physiological Society and The Physiological Society.
A. A. Zeki et al.
Inhaled Statin for the Treatment of Asthma
A
OVA + i.t.PBS
OVA + i.t. pravastatin
FA + i.t. PBS
FA + i.t. pravastatin
B
(n = 3)
(n = 6)
(n = 6)
(n = 3)
Figure 6. Effect of Intratracheal Pravastatin on Bronchial Epithelium and Mouse Weights. (A) I.t. pravastatin did not damage the airway
epithelium as seen by histological assessment (H&E stain, 4009 magnification). (B) I.t. pravastatin in both FA controls and OVA-exposed mice
was well-tolerated as measured by steady weight gain in all mice (P = NS by 2-way ANOVA). There were no statistically significant weight
differences between groups on Day #1, that is, at their baseline weights (P = NS by 1-way ANOVA). Compared to their own baseline, mice in
each group gained weight steadily over time during drug treatment. The number of mice (n) is indicated in parentheses for each treatment
group.
achieve similar anti-inflammatory effects in the lung,
while reducing systemic absorption and unwanted potential side effects. In some patients, statins can have serious
adverse effects including hepatitis and myositis that would
preclude their clinical use. Even more common yet milder
symptoms of statin use such as myalgias could potentially
by bypassed if minimal systemic absorption can be
achieved. This might open new therapeutic avenues for
pediatric patients with asthma, where statins are not routinely used, or in elderly patients intolerant to statin side
effects such as myalgias. Based on biological plausibility
(Yeganeh et al. 2014), an inhaled statin may have the
potential to mitigate adverse airway remodeling (Murphy
et al. 2008; Zeki et al. 2010; Ahmad et al. 2011), in
particular goblet cell mucus production (Marin et al.
2013), smooth muscle hyperplasia (Takeda et al. 2006;
Schaafsma et al. 2011), subepithelial fibrosis (Watts and
Spiteri 2004; Watts et al. 2005; Kim et al. 2010), and
extracellular matrix (ECM) production (Li et al. 2008;
Schaafsma et al. 2011); where we currently lack adequate
therapy.
Summary of main findings
Pravastatin given by i.t. instillation achieves high relative
concentrations in BALF and lung relative to plasma, as
measured by mass spectrometry (Fig. 1). In comparing the
effect of OVA relative to FA controls, pravastatin achieves
the highest distribution in lung tissue (greater than BALF
or plasma), indicating little but detectible systemic
absorption (Fig. 2). While pravastatin reduced goblet cell
metaplasia/hyperplasia (Fig. 3), we saw no statistically
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2015 | Vol. 3 | Iss. 5 | e12352
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A. A. Zeki et al.
Inhaled Statin for the Treatment of Asthma
significant anti-inflammatory effects besides reductions in
select cytokines (TNFa and KC) (Fig. 5). Importantly, pravastatin reduced AHS and total Rrs, but did not attenuate
AHR or preserve lung compliance (Fig. 4). Finally, pravastatin administered i.t. was well-tolerated in mice and did
not damage the airway epithelium (Fig. 6).
There is only one other study that we are aware of
which systematically evaluated the impact of simvastatin
in murine allergic asthma via multiple routes including
by gavage, intraperitoneal injection, intratracheal instillation, and aerosol inhalation; where the authors also
reported simvastatin levels in lung and blood (Xu et al.
2012). In this study, they found that intratracheal and
inhaled simvastatin had potent anti-inflammatory effects
similar to the corticosteroid dexamethasone, and significantly improved both AHR and lung compliance. However, the use of 20% ethanol as simvastatin’s drug vehicle
raises great concern given the potential cytotoxic effects
of alcohol. This high ethanol concentration may preclude
use in humans and the rapid translation to a Phase 1
clinical trial, despite the recent development of a novel
statin inhaler for human use (Tulbah et al. 2014).
Novel method for measuring pravastatin in
plasma and lung tissues
We used UPLC-MS to measure pravastatin drug concentrations in plasma, lung tissue, and BALF (Fig. 1).
Although pravastatin has been measured in plasma previously (Jain et al. 2007; Deng et al. 2008; Badolo et al.
2013), the use of mass spectrometry to measure statins in
lung tissue has not been previously reported. More specifically, the use of UPLC-MS to quantify statin drug levels,
including pravastatin, in lung tissues is novel and unique
with respect to the known published literature.
We found that pravastatin remained largely in the
BALF and lung tissues, and there are numerous factors
that could contribute to this outcome. Because we only
examined a single time point after pravastatin i.t. instillation, this observation could be a function of the timing
of pravastatin administration relative to the time mice
were killed (Fig. 1A–C). Specimens were collected at the
end of 2-week experiments within a 1 to 2 h window
after the last OVA exposure/statin dose. For instance, we
did not measure pravastatin in plasma, lung, or BALF at
the potential nadir between dosing episodes where the
partitioning of pravastatin between these three tissue
compartments could have been different (i.e., more equal
drug distribution between lung and blood). On the basis
of pravastatin plasma concentrations at the prespecified
time points (see Materials and Methods), we concluded
that minimal yet readily detectible systemic absorption
occurred.
2015 | Vol. 3 | Iss. 5 | e12352
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Normal lung clearance of the drug may include contributions via the mucociliary escalator, which would result
in the cleared drug being swallowed, leading to secondary
and indirect systemic administration. Distribution pathways from airways to plasma for pravastatin administered
via the intratracheal route could include direct diffusion
through the airway endothelium into the bloodstream,
lymphatic drainage, or swallowing of lung lining fluid
during mucociliary clearance.
Xu et al. (2012) compared intratracheally administered
and inhaled simvastatin in their allergic asthma model.
The investigators used HPLC to measure simvastatin in
different tissue compartments. Similar to our results, they
reported much higher simvastatin levels in the lung as
compared to plasma measured at 0.5, 2, and 6 h after
injection, with the i.t. route achieving much higher levels
than aerosol inhalation (186.3 i.t. vs. 6.85 inhaled (lg/g
tissue)). This confirms our hypothesis that the majority
of the drug remains in the local pulmonary tissue,
whereas only a relatively small amount is absorbed systemically at the time range observed.
We also evaluated the effect of allergic inflammation
on the partitioning of pravastatin between the plasma,
lung, and BALF compartments (Fig. 2). After normalizing
lung and BALF pravastatin levels to each animal’s own
plasma pravastatin concentration, we obtained ‘corrected’
or normalized relative pravastatin values (see Materials
and Methods and Results). This allowed us to correct for
any potential variation from mouse to mouse in relative
systemic absorption of the drug. Then we compared these
normalized pravastatin levels as ratios of OVA-to-FA, and
observed that during OVA-induced inflammation, pravastatin levels are 4.73-fold higher in the lung but only
2.86-fold higher in BALF as compared to FA controls;
and with stable levels in plasma (Fig. 2B).
These data suggest that during inflammation, more
pravastatin enters (or remains) in the lung tissue as compared to BALF. We speculate that under conditions of
allergic inflammation, epithelial-vascular leaking may
underlie the higher pravastatin drug levels found in the
lung compared to BALF. Surprisingly, plasma drug levels
between OVA and FA groups did not change by comparison of either drug concentrations (Fig. 1B and C) or normalized ratios (see Results section). Despite a higher drug
concentration in the lung as compared to BALF or
plasma, pravastatin’s lack of significant anti-inflammatory
effects on leukocyte influx is unexpected.
Pravastatin effects on airway epithelial
remodeling
Pravastatin’s most potent effect was on reducing airway
goblet cell metaplasia/hyperplasia (Fig. 3). Direct instilla-
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the American Physiological Society and The Physiological Society.
A. A. Zeki et al.
tion of pravastatin into mouse airways attenuated OVAinduced goblet cell hyperplasia by approximately one
third, an effect that is comparable to that seen with systemic simvastatin treatment (Kim et al. 2007; Zeki et al.
2010). This finding is not surprising given that statins are
known to have direct effects on airway epithelial cells
(Sakoda et al. 2006; Murphy et al. 2008; Takahashi et al.
2008; Planaguma et al. 2010; Xing et al. 2011; Iwata et al.
2012; Zeki et al. 2012; Brandelius et al. 2013; Lee et al.
2013), including simvastatin which reduces mucus production in Calu-3, a human airway epithelial cell line
(Marin et al. 2013). In vivo application of inhaled simvastatin also reduced airway goblet cell remodeling and
mucus production (Xu et al. 2012) similar to our results
with i.t. pravastatin (Fig. 3).
The Muc5AC gene is one of several key mucin genes
that programs epithelial cell goblet cell differentiation and
mucin production. Simvastatin treatment by the intraperitoneal route reduces Muc5AC mRNA in rat lungs
(Ou et al. 2008). Similarly, intragastric administration of
simvastatin also reduces Muc5AC mucin synthesis at both
the mRNA and protein synthesis levels in rat lungs (Chen
et al. 2010).
Whether statins have direct effects on Muc5AC gene
expression remains an open question. Because IL13
induces Muc5AC expression in human airway epithelial
cells (Zhao et al. 2009), and statins are known to inhibit
IL13 production in lungs (Zeki et al. 2009), we speculate
that this could be one potential mechanism of how statins
attenuate mucin production in airways. Because overproduction of mucus in humans is found in 80% of lethal
cases of severe asthma, pravastatin’s direct effect on goblet
cells is evidence supporting the potential benefits of developing statin inhalers.
Pravastatin inhibition of goblet cell hyperplasia/metaplasia may be independent of inflammation given that
pravastatin did not inhibit airway leukocyte influx in
BALF to a significant degree. We speculate that pravastatin’s beneficial effects via the i.t. route may be predominantly limited to the airway epithelial lining at the dose
used (30 mg/kg). Additional research is needed to examine whether or not this is a sustained phenomenon or
something related to our experimental protocol, dosing,
and timing of drug administration.
Pravastatin effects on inflammation
Despite the fact that i.t. administration of pravastatin
achieved appreciably high concentrations in BALF and
lung tissue (Figs. 1, 2), pravastatin did not consistently
reduce inflammatory cell influx as measured in BALF.
Although we observed reduced peribronchiolar inflammation by H&E staining (Fig. 5D) and inhibition of TNFa
Inhaled Statin for the Treatment of Asthma
and KC in BALF (Fig. 5A and B), the lack of statistically
significant anti-inflammatory effects of pravastatin on
BALF cell counts (Fig. 5C and Table 1) was a surprise
given what is known about statins in general, and what
we know about pravastatin in the OVA model (Yeh and
Huang 2004; Imamura et al. 2009). Furthermore, it
remains unclear whether i.t. pravastatin affects inflammatory cell migration from the peribronchial region into the
airway lumen or tissue adherence which may affect the
visual impressions of our lung histology results as seen in
Fig. 5D.
There may be several reasons for this unexpected
result. First, unlike the more lipophilic statins such as
simvastatin, fluvastatin, and atorvastatin; pravastatin is
the most hydrophilic statin available and therefore does
not enter cells via passive diffusion. Therefore, it is possible that while pravastatin is in the lung tissue, it is unable
to efficiently enter airway resident cells such as the
epithelium or inflammatory/immune cells. Pravastatin
requires specific organic anion transporting polypeptides
in order to pass through the cell plasma membrane, and
it remains unknown if lung or airway cells express these
transporters (see below). Second, pravastatin’s effect
could have been short lived and therefore missed during
the time points we evaluated. Third, pravastatin’s beneficial effects with respect to anti-inflammatory potency
may be limited to immune cell and endothelial cell functions, rather than to airway epithelial or mesenchymal
cells. Fourth, the concentration we used may not have
been sufficient to inhibit inflammation; however, the pravastatin dose we used (30 mg/kg) is the maximal concentration where the drug remains soluble in aqueous
solutions. Fifth, pravastatin may be more effective via systemic routes (i.e., i.p. or oral) (Yeh and Huang 2004;
Imamura et al. 2009), rather than via inhalation or direct
airway instillation. And finally, it is possible that a
chronic OVA exposure model (i.e., 8 or 10 weeks) might
yield different results with respect to anti-inflammatory
and antifibrotic effects of pravastatin. Additional investigations are needed in order to answer these important
questions.
Pravastatin effects on pulmonary mechanics
On the basis of known beneficial effects of statins on lung
physiology (Chiba et al. 2008a,b; Zeki et al. 2009; Cazzola
et al. 2011; Xu et al. 2012), we predicted that i.t. pravastatin would have potent effects on airway resistance and
lung compliance. Instead, we found that pravastatin had
differential effects on airway mechanics. Despite reducing
total Rrs (Fig. 4B) in OVA-exposed mice, pravastatin
overall did not improve lung compliance or attenuate
MCh-induced AHR (Fig. 4A and D).
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
2015 | Vol. 3 | Iss. 5 | e12352
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A. A. Zeki et al.
Inhaled Statin for the Treatment of Asthma
However, treatment with i.t. pravastatin did reduce
AHS, a clinically relevant component of airway hyperresponsiveness. Pravastatin-treated animals required MCh
doses twice as high as the PBS control group to achieve
an equivalent increase in Rrs above baseline (Fig. 4C).
These results indicate that pravastatin’s protective effect
on airway resistance is limited to the sensitivity of airways
to broncho-constricting agents. This translates to higher
broncho-provocating MCh doses needed to achieve a
given increase in Rrs in statin treated mice, indicating a
protective effect of pravastatin. However, once this sensitivity threshold is surpassed then pravastatin offers no
additional mitigating effects on AHR or bronchospasm
(O’Byrne and Inman 2003; Affonce and Lutchen 2006;
Turi et al. 2011).
There are several potential reasons for this uncoupling
of AHR and AHS effects due to pravastatin treatment. It
may be that pravastatin did not reach airway smooth
muscles and instead remained on the luminal epithelial
side or only within the epithelial cells, thereby having no
effect on AHR. Another potential explanation is direct
effects on airway neuronal cells or their conductive function (Tarhzaoui et al. 2009), thereby affecting the neural
contribution to bronchoconstriction. Of note, different
agonists or pharmacologic agents can induce changes in
both AHR and AHS. However, they can also affect either
AHR or AHS independently, yielding specific effects on
only one component of airways hyperresponsiveness
(L€
otvall et al. 1998; Chapman et al. 2014).
Statin physiochemical properties and their
anti-inflammatory effects
The physiochemical properties of statins may have significant effects on the absorption and activation of statins
in vivo, determining both transport inside cells and their
conversion to the hydroxy acid, the active form of statin
that binds HMGCR (Hamelin and Turgeon 1998; Istvan
and Deisenhofer 2001). Statin polarity (i.e., hydrophilicity
vs. hydrophobicity) may impact statin tissue absorption
and distribution, and thereby its ultimate anti-inflammatory potential.
Simvastatin is the most lipophilic of the statins used
clinically, whereas pravastatin is the most hydrophilic.
While these properties of polarity inform experimental
design with respect to drug solubility and delivery, they
may be equally pertinent for cell penetration, and thus,
statin bioavailability, potency, and efficacy. The active
form of statins is the ‘open ring’ hydroxy acid (e.g., simvastatin acid) which binds to the active site of HMGCR
to inhibit its enzymatic function. Simvastatin is administered as an inactive prodrug (‘closed ring’ lactone).
Enzymes such as serum lactonases and paraoxonases
2015 | Vol. 3 | Iss. 5 | e12352
Page 16
(Draganov et al. 2000), alkaline hydrolases (Hamelin and
Turgeon 1998), and carboxylesterases open the ring to
produce the active form of simvastatin. These enzymes
also hydrolyze other statins besides simvastatin.
Unlike simvastatin, pravastatin is administered as the
active hydroxy acid. However, this active form of statins
is not as readily absorbed by cells as is the inactive form.
Intracellular uptake of pravastatin requires organic anion
transporting polypeptide (OATP) cell membrane transporters which are variably found in peripheral tissues outside the liver. Whether OATPs are expressed in normal
human lungs and specifically airway epithelial cells is not
known. By comparison, simvastatin enters cells by passive
cell membrane diffusion given its lipophilicity, and is
more likely than pravastatin to enter the systemic circulation and reach extra-hepatic tissues (Kleemann and
Kooistra 2005).
Among the statins, pravastatin is the most hydrophilic
with a partition coefficient (Log D) of 0.84 at a pH of
7.4 (Mctaggart 2003) (or 0.23 at a pH of 7.0) (Serajuddin
et al. 1991). Although the hydrophilicity of oral pravastatin allows for relatively high circulating concentrations, it
also hinders penetration to peripheral tissues and prevents
passive diffusion into cells (Hamelin and Turgeon 1998;
Vaughan and Gotto 2004).
Cellular uptake of pravastatin is dependent on carriermediated transport proteins, primarily OATP2 and
OATP1B1 (a.k.a. OATP-C) in humans, which are abundant in hepatocytes but not known to be expressed in
lungs. However, there is variable expression in other
peripheral tissues such as human brain, kidney, liver,
intestines, testis, placenta, heart, and skin (Hsiang et al.
1999; Cheng et al. 2005; Kalliokoski and Niemi 2009).
Other pravastatin transporters include OATP1B3 (Seithel
et al. 2007) and OATP2B1 (Nozawa et al. 2004; Kalliokoski and Niemi 2009). Mouse OATP expression is highly
variable in different tissues, but is expressed in mouse
lungs (OATP2a1, OATP3a1, OATP4c1, OATP5a1)
(Cheng et al. 2005); however, it remains unknown
whether these specific forms of OATP transport pravastatin. Rat OATP1 and OATP2 transport pravastatin, but
they are not expressed in lung tissue (Noe et al. 1997;
Hsiang et al. 1999; Hasegawa et al. 2010). Whereas,
OATP3 is expressed in lung tissue in rats, but there is no
report of pravastatin transport capability (Walters et al.
2000; Ohtsuki et al. 2004).
Therefore, while OATPs are expressed at relatively high
levels in murine lungs, we do not know whether they can
transport pravastatin across the epithelial cell membrane.
Although pravastatin was administered directly to the airways in our studies, the lack of a mechanism to readily
enter cells may have prevented pravastatin from reaching
high enough intracellular concentrations to inhibit
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
A. A. Zeki et al.
HMGCR by any appreciable amount, thus providing little
to no therapeutic anti-inflammatory effect, despite beneficial effects on goblet cells and AHS. However, our experimental design also does not exclude other potential
pleiotropic effects of pravastatin. Future studies should
measure the expression of OATPs in lungs, including in
airway epithelial cells, in order to test this hypothesis.
Conversely, simvastatin is 173 times more lipophilic
than pravastatin (Log P of 4.68) in the lactone form, and
195 times more lipophilic than pravastatin in the hydroxy
acid form (at a pH of 7.0) (Serajuddin et al. 1991), and is
therefore capable of passively diffusing through cell membranes without the need for anionic transporters (Serajuddin et al. 1991; Hamelin and Turgeon 1998; Schachter
2005). However, the bioavailability of oral simvastatin is
only 5%, primarily due to low solubility (95–98% protein
bound) and an efficient first-pass metabolism (Corsini
et al. 1999; Vaughan and Gotto 2004; Schachter 2005;
Pandya et al. 2008). By bypassing this first-pass metabolism through the administration of low dose simvastatin
via the intratracheal route, potent anti-inflammatory
effects in the airways are observed (Xu et al. 2012). Passive diffusion may allow intracellular simvastatin levels to
reach high enough concentrations to provide the myriad
of beneficial effects predicted but not observed in our
study using pravastatin.
The ability to passively diffuse through cell membranes
may also allow simvastatin to escape from lung tissue to
the systemic circulation at a higher rate than pravastatin.
This might prevent potential statin-induced toxicities
caused by an over-accumulation of the drug in lung tissue. However, higher systemic absorption of simvastatin
compared to pravastatin may also result in higher systemic side effects. Xu et al. (2012) did not report any toxicity or adverse reactions with i.t. or inhaled simvastatin
(using 20% ethanol as drug vehicle). By comparison, the
inability of pravastatin to freely cross cell membranes may
also inhibit it from exiting the lung compartment to enter
the systemic circulation, potentially allowing the drug to
concentrate in the airways or lung tissue and increase the
risk of potential statin-induced toxicities (Schachter
2005). In our experiments, we did not observe any pravastatin related lung or systemic toxic effects (Fig. 6).
Additional research is required to answer these important
questions given their therapeutic and safety implications.
We speculate that while i.t. pravastatin is a logical and
pragmatic choice given its translational potential in
human asthma, its physiochemical properties likely contributed to low intracellular levels despite being found in
high concentrations in lung tissue homogenate and
BALF. While it is possible that the pulmonary antiinflammatory effect of statins may be due to systemic
(Imamura et al. 2009; Zeki et al. 2009) rather than local-
Inhaled Statin for the Treatment of Asthma
ized effects in the lung, the work by Xu et al. using simvastatin does not support this idea. In our experiments,
low-level systemic absorption of pravastatin was not adequate enough to significantly reduce lung or airway
inflammation (Figs. 1, 5). However, a different and more
lipophilic statin such as simvastatin could potentially
produce the desired effects via inhalation in human
asthma.
Alternative methods of administering
statins
At present, the only approved route of statin drug administration in humans is through oral ingestion. There are
many benefits to administering statins orally, such as ease
of administration, good patient compliance, minimal sterility constraints, and cost-effectiveness. However, there
are also some drawbacks to oral administration. For
example, the bioavailability and the general circulation of
statins are relatively low due to first-pass metabolism and
protein binding (Hamelin and Turgeon 1998; Vaughan
and Gotto 2004). This in part depends on the extent of
binding to plasma proteins and cell permeability, and
these factors depend on the physiochemical properties of
the statin in question. Research is needed to develop better and more efficient ways to administer statin drugs that
can increase their bioavailability in specific tissue compartments or organs, such as the airways or lungs.
Other potential routes for statin delivery include nasal
(Jha et al. 2014), intravenous (Prinz et al. 2008), subcutaneous (Bews et al. 2014; Jha et al. 2014), intratracheal
nanoparticle (Chen et al. 2011), nebulized formulations for
direct inhalation, and controlled-release statin-loaded
microspheres (Kanjickal et al. 2004; Vishwanathan 2008).
Nanocarriers are also a potential innovative drug vehicle,
and in particular, novel formulations that can deposit and
concentrate in the lung before releasing their statin payload
in order to achieve higher local steady-state drug levels.
Our study is the first to evaluate the potential of i.t.
pravastatin in asthma and the second to evaluate statin
administration via the intratracheal route (Xu et al.
2012). There are several potential benefits of administering statins via inhalation and a rationale to support this
therapeutic approach: Lower effective dose as compared
to systemic treatment, reduced systemic absorption with
attendant lower systemic side effects, direct effect on airway epithelial cells, potential treatment for airway smooth
muscle hypertrophy/hyperplasia, potential use in pediatric
patients, an alternative option for patients for whom oral
statins are contraindicated due to myopathy or other
adverse effects, and combination therapy with other standard-of-care inhaler medications, such as ICS, LABA,
LAMA.
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
2015 | Vol. 3 | Iss. 5 | e12352
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A. A. Zeki et al.
Inhaled Statin for the Treatment of Asthma
Study limitations
There are several limitations to our study. While plasma
and BALF pravastatin concentrations were directly measured and quantified in units of ng/lL, lung pravastatin
concentration was estimated based on an approximation
of lung density. This allowed for relative comparisons to
be made across three major tissue compartments. However, our results should be interpreted with this limitation
in mind; these are, at best, relative comparisons. Additional calculations using rat lung densities gave us similar
results (data not shown). We therefore determined that
an estimate of lung pravastatin concentration is a reasonable approximation for the necessary comparisons to
BALF and plasma pravastatin levels. In the Results section, we also report the exact drug concentration per lung
and per gram of lung.
A given volume of plasma, BALF, and lung tissue may
not be the same with respect to the distribution of pravastatin, or any drug for that matter. However, because pravastatin is highly water soluble, we assumed that pravastatin
was in equilibrium with total body water. If this assumption is true, we can then directly compare pravastatin concentrations between all three tissue compartments (in units
of ng/lL, Fig. 1). Therefore, any conclusions drawn from
these data should be tempered by this potential limitation.
We did not measure pravastatin concentrations at different time points, thereby excluding the nadir levels on
days between statin treatments. This could have yielded
different results with respect to the equilibrium achieved
between the three tissue compartments; BALF, lung, and
plasma. Thus, it is possible that relative pravastatin levels
would have differed from what we observed in Figs. 1, 2.
However, given similar findings by Xu et al. using simvastatin at earlier time points (Xu et al. 2012), we believe
our results are valid and pertinent.
With respect to pravastatin’s effect on lung physiology,
we recognize that our use of invasive plethysmography
yields limited information as compared to the more sensitive forced oscillation technique (FOT). While we were able
to detect statin-mediated beneficial effects on AHS, other
more specific effects on airway narrowing, peripheral tissue
resistance, and tissue elastance typically assessed using FOT
were not measured using our technique. Therefore, it is
possible that additional statin effects on airway or lung
function remain undiscovered. Future work in this field
using FOT could yield additional important results worthy
of further study.
Conclusions
Our data demonstrate the ability to directly measure and
detect pravastatin in lung tissues using mass spectrometry,
2015 | Vol. 3 | Iss. 5 | e12352
Page 18
and the ability of the drug to concentrate in the lung
while achieving minimal systemic absorption. Our results
also indicate that intratracheal pravastatin has the potential to mitigate some pathological features of experimental
allergic asthma. While anti-inflammatory effects were
modest at best, i.t. pravastatin reduced the production of
select cytokines, attenuated goblet cell epithelial remodeling, and reduced AHS. Finally, pravastatin applied
directly to airways in vivo appeared to be safe and welltolerated by mice.
The use of UPLC-MS allowed us to measure statins in
any tissue, in particular the lungs. This will be an important aspect of translating findings from animal models to
humans, as the need to quantify statins in varied tissues
remains relevant in the human host as we explore alternative routes of statin delivery.
Our results indicate that the statins should be explored
as a potential novel class of inhaler therapy for airway diseases such as asthma. However, we believe that additional
preclinical work is needed to determine the optimal statin
dose, route of administration (and safety of inhalation
long-term), and mechanism(s) involved in order to guide
future translational studies. We predict that statins
with greater lipophilicity may yield more potent antiinflammatory effects than pravastatin. Work in this area of
“lung-directed” or inhaled statins is a new chapter in the
evolution of these drugs for the treatment of lung diseases.
Conflict of Interest
None declared.
References
Affonce, D. A., and K. R. Lutchen. 2006. New perspectives
on the mechanical basis for airway hyperreactivity and
airway hypersensitivity in asthma. J. Appl. Physiol.
101:1710–1719.
Ahmad, T., U. Mabalirajan, A. Sharma, J. Aich, L. Makhija,
B. Ghosh, et al. 2011. Simvastatin improves epithelial
dysfunction and airway hyperresponsiveness: from
asymmetric dimethyl-arginine to asthma. Am. J. Respir. Cell
Mol. Biol. 44:531–539.
Alexeeff, S. E., A. A. Litonjua, D. Sparrow, P. S. Vokonas, and
J. Schwartz. 2007. Statin use reduces decline in lung
function: VA Normative Aging Study. Am. J. Respir. Crit.
Care Med. 176:742–747.
Badolo, L., C. Bundgaard, M. Garmer, and B. Jensen. 2013.
The role of hepatic transport and metabolism in the
interactions between pravastatin or repaglinide and two
rOatp inhibitors in rats. Eur. J. Pharm. Sci. 49:767–772.
Bews, H. J., J. C. Carlson, A. Jha, S. Basu, A. J. Halayko, and
C. S. Wong. 2014. Simultaneous quantification of
simvastatin and simvastatin hydroxy acid in blood serum at
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
A. A. Zeki et al.
physiological pH by ultrahigh performance liquid
chromatography-tandem mass spectrometry (UHPLC/MS/
MS). J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
947–948:145–150.
Braganza, G., R. Chaudhuri, C. McSharry, C. J. Weir, I.
Donnelly, L. Jolly, et al. 2011. Effects of short-term
treatment with atorvastatin in smokers with asthma–a
randomized controlled trial. BMC Pulm. Med. 11:16.
Brandelius, A., I. Mahmutovic Persson, J. Calven, L. Bjermer,
C. G. Persson, M. Andersson, et al. 2013. Selective
inhibition by simvastatin of IRF3 phosphorylation and TSLP
production in dsRNA-challenged bronchial epithelial cells
from COPD donors. Br. J. Pharmacol. 168:363–374.
Capra, V., and G. E. Rovati. 2014. Rosuvastatin inhibits
human airway smooth muscle cells mitogenic response to
eicosanoid contractile agents. Pulm. Pharmacol. Ther.
27:10–16.
Cazzola, M., L. Calzetta, C. P. Page, B. Rinaldi, A. Capuano,
and M. G. Matera. 2011. Protein prenylation contributes to
the effects of LPS on EFS-induced responses in human
isolated bronchi. Am. J. Respir. Cell Mol. Biol. 45:
704–710.
Chapman, D. G., J. E. Tully, J. D. Nolin, Y. M. JanssenHeininger, and C. G. Irvin. 2014. Animal models of allergic
airways disease: where are we and where to next? J. Cell.
Biochem. 115:2055–2064.
Chen, Y. J., P. Chen, H. X. Wang, T. Wang, L. Chen, X.
Wang, et al. 2010. Simvastatin attenuates acrolein-induced
mucin production in rats: involvement of the Ras /
extracellular signal-regulated kinase pathway. Int.
Immunopharmacol. 10:685–693.
Chen, L., K. Nakano, S. Kimura, T. Matoba, E. Iwata, M.
Miyagawa, et al. 2011. Nanoparticle-mediated delivery of
pitavastatin into lungs ameliorates the development and
induces regression of monocrotaline-induced pulmonary
artery hypertension. Hypertension 57:343–350.
Cheng, X., J. Maher, C. Chen, and C. D. Klaassen. 2005.
Tissue distribution and ontogeny of mouse organic anion
transporting polypeptides (OATPS). Drug Metab. Dispos.
33:1062–1073.
Chiba, Y., J. Arima, H. Sakai, and M. Misawa. 2008a.
Lovastatin inhibits bronchial hyperresponsiveness by
reducing RhoA signaling in rat allergic asthma. Am. J.
Physiol. Lung Cell. Mol. Physiol. 294:L705–L713.
Chiba, Y., S. Sato, and M. Misawa. 2008b. Inhibition of
antigen-induced bronchial smooth muscle
hyperresponsiveness by lovastatin in mice. J. Smooth Muscle
Res. 44:123–128.
Corsini, A., S. Bellosta, R. Baetta, and R. Fumagalli. 1999. New
insights into the pharmacodynamic and pharmacokinetic
properties of statins. Pharmacol. Ther. 84:413–428.
Cowan, D. C., J. O. Cowan, R. Palmay, A. Williamson, and D.
R. Taylor. 2010. Simvastatin in the treatment of asthma:
lack of steroid-sparing effect. Thorax 65:891–896.
Inhaled Statin for the Treatment of Asthma
Davis, B. B., A. A. Zeki, J. M. Bratt, L. Wang, S. Filosto,
W. F. Walby, et al. 2013. Simvastatin inhibits smokeinduced airway epithelial injury: implications for COPD
therapy. Eur. Respir. J. 42:350–361.
Deng, J. W., K. B. Kim, I. S. Song, J. H. Shon, H. H. Zhou, K.
H. Liu, et al. 2008. Determination of two HMG-CoA
reductase inhibitors, pravastatin and pitavastatin, in plasma
samples using liquid chromatography – tandem mass
spectrometry for pharmaceutical study. Biomed.
Chromatogr. 22:131–135.
Draganov, D. I., P. L. Stetson, C. E. Watson, S. S. Billecke, and
B. N. La Du. 2000. Rabbit serum paraoxonase 3 (PON3) is
a high density lipoprotein-associated lactonase and protects
low density lipoprotein against oxidation. J. Biol. Chem.
275:33435–33442.
Fahimi, F., J. Salamzadeh, H. Jamaati, S. Sohrabi, A.
Fakharian, and Z. Mohammadtaheri. 2009. Do statins
improve lung function in asthmatic patients? A randomized
and double-blind trial. Iran J. Pharm. Sci. 5:13–20.
Greenwood, J., L. Steinman, and S. S. Zamvil. 2006. Statin
therapy and autoimmune disease: from protein prenylation
to immunomodulation. Nat. Rev. Immunol. 6:358–370.
Hamelin, B. A., and J. Turgeon. 1998. Hydrophilicity /
lipophilicity: relevance for the pharmacology and clinical
effects of HMG- CoA reductase inhibitors. Trends
Pharmacol. Sci. 19:26–37.
Hasegawa, Y., S. Kishimoto, N. Shibatani, and N. Inotsume.
2010. The disposition of pravastatin in a rat model of
streptozotocin-induced diabetes and Organic anion
transporting polypeptide 2 and multidrug resistanceassociated protein 2 expression in the liver. Biol. Pharm.
Bull. 33:153–156.
Hothersall, E. J., R. Chaudhuri, C. McSharry, I. Donnelly, J.
Lafferty, A. D. McMahon, et al. 2008. Effects of atorvastatin
added to inhaled corticosteroids on lung function and
sputum cell counts in atopic asthma. Thorax 63:1070–1075.
Hsiang, B., Y. Zhu, Z. Wang, Y. Wu, V. Sasseville, W. P. Yang,
et al. 1999. A novel human hepatic organic anion
transporting polypeptide (OATP2). Identification of a liverspecific human organic anion transporting polypeptide and
identification of rat and human hydroxymethylglutaryl-CoA
reductase inhibitor transporters. J. Biol. Chem. 274:37161–
37168.
Huang, C.-C., W.-L. Chan, Y.-C. Chen, T. J. Chen, K. T.
Chou, S. J. Lin, et al. 2011. Statin use in patients with
asthma: a nationwide population-based study. Eur. J. Clin.
Invest. 41:507–512.
Huang, C.-F., H.-J. Peng, C.-C. Wu, W.-T. Lo, Y.-L. Shih, and
T.-C. Wu. 2013. Effect of oral administration with
pravastatin and atorvastatin on airway hyperresponsiveness
and allergic reactions in asthmatic mice. Ann. Allergy
Asthma Immunol. 110:11–17.
Imamura, M., K. Okunishi, H. Ohtsu, K. Nakagome, H.
Harada, R. Tanaka, et al. 2009. Pravastatin attenuates
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
2015 | Vol. 3 | Iss. 5 | e12352
Page 19
A. A. Zeki et al.
Inhaled Statin for the Treatment of Asthma
allergic airway inflammation by suppressing antigen
sensitization, interleukin 17 production and antigen
presentation in the lung. Thorax 64:44–49.
Istvan, E. S., and J. Deisenhofer. 2001. Structural mechanism
for statin inhibition of HMG-CoA reductase. Science
292:1160–1164.
Iwata, A., R. Shirai, H. Ishii, H. Kushima, S. Otani, K.
Hashinaga, et al. 2012. Inhibitory effect of statins on
inflammatory cytokine production from human bronchial
epithelial cells. Clin. Exp. Immunol. 168:234–240.
Jain, D. S., G. Subbaiah, M. Sanyal, V. K. Jain, and P.
Shrivastav. 2007. A rapid and specific approach for direct
measurement of pravastatin concentration in plasma by LCMS / MS employing solid-phase extraction. Biomed.
Chromatogr. 21:67–78.
Jha, A., S. Basu, M. Ryu, O. Ojo, J. Schwartz, and A. J.
Halayko. 2014. Inhibition of airway inflammation and
hyperreactivity by inhaled simvastatin. Am. J. Respir. Crit.
Care Med. 189:A2690. (abstract).
Joyce, M., C. J. Kelly, G. Chen, and D. J. Bouchier-Hayes.
2001. Pravastatin attenuates lower torso ischaemiareperfusion induced lung injury by upregulating constitutive
endothelial nitric oxide synthase. Eur. J. Vasc. Endovasc.
Surg. 21:295–300.
Kalliokoski, A., and M. Niemi. 2009. Impact of OATP
transporters on pharmacokinetics. Br. J. Pharmacol.
158:693–705.
Kanjickal, D., S. Lopina, M. M. Evancho-Chapman, S.
Schmidt, D. Donovan, and S. Springhetti. 2004. Polymeric
sustained local drug delivery system for the prevention of
vascular intimal hyperplasia. J. Biomed. Mater. Res. A
68:489–495.
Kenyon, N. J., R. W. Ward, and J. A. Last. 2003. Airway
fibrosis in a mouse model of airway inflammation. Toxicol.
Appl. Pharmacol. 186:90–100.
Kim, D., J. Lim, Y. Lee, J. Ro, and S. Ryu. 2007. Antiinflammatory mechanism of simvastatin in mouse allergic
asthma model. Eur. J. Pharmacol. 557:76–86.
Kim, J. W., C. K. Rhee, T. J. Kim, Y. H. Kim, S. H. Lee, H. K.
Yoon, et al. 2010. Effect of pravastatin on bleomycininduced acute lung injury and pulmonary fibrosis. Clin.
Exp. Pharmacol. Physiol. 37:1055–1063.
Kleemann, R., and T. Kooistra 2005. HMG-CoA reductase
inhibitors: effects on chronic subacute inflammation and
onset of atherosclerosis induced by dietary cholesterol.
Curr. Drug Targets Cardiovasc. Haematol. Disord. 5:
441–453.
Krauth, M. T., Y. Majlesi, K. Sonneck, P. Samorapoompichit,
M. Ghannadan, A. W. Hauswirth, et al. 2006. Effects of
various statins on cytokine-dependent growth and IgEdependent release of histamine in human mast cells. Allergy
61:281–288.
Lee, C. S., E. H. Yi, J.-K. Lee, C. Won, Y. J. Lee, M. K. Shin,
et al. 2013. Simvastatin suppresses RANTES-mediated
2015 | Vol. 3 | Iss. 5 | e12352
Page 20
neutrophilia in polyinosinic-polycytidylic acid-induced
pneumonia. Eur. Respir. J. 41:1147–1156.
Li, M., Z. Li, and X. Sun. 2008. Statins suppress MMP2
secretion via inactivation of RhoA/ROCK pathway in
pulmonary vascular smooth muscles cells. Eur. J. Pharmacol.
591:219–223.
L€
otvall, J., M. Inman, and P. O’Byrne. 1998. Measurement of
airway hyperresponsiveness: new considerations. Thorax
53:419–424.
Maneechotesuwan, K., W. Ekjiratrakul, K. Kasetsinsombat, A.
Wongkajornsilp, and P. J. Barnes. 2010. Statins enhance the
anti-inflammatory effects of inhaled corticosteroids in
asthmatic patients through increased induction of
indoleamine 2, 3-dioxygenase. J. Allergy Clin. Immunol.
126:754–762.e1.
Marin, L., D. Traini, M. Bebawy, P. Colombo, F. Buttini,
M. Haghi, et al. 2013. Multiple dosing of simvastatin
inhibits airway mucus production of epithelial cells:
implications in the treatment of chronic obstructive airway
pathologies. Eur. J. Pharm. Biopharm. 84:566–572.
McKay, A., B. P. Leung, I. B. McInnes, N. C. Thomson, and F.
Y. Liew. 2004. A novel anti-inflammatory role of simvastatin
in a murine model of allergic asthma. J. Immunol.
172:2903–2908.
Mctaggart, F. 2003. Comparative pharmacology of
rosuvastatin. Atheroscler. Suppl. 4:9–14.
Melo, A. C., S. S. Valencßa, L. B. Gitirana, J. C. Santos, M. L.
Ribeiro, M. N. Machado, et al. 2013. Redox markers and
inflammation are differentially affected by atorvastatin,
pravastatin or simvastatin administered before endotoxininduced acute lung injury. Int. Immunopharmacol. 17:57–64.
Moini, A., G. Azimi, and A. Farivar. 2012. Evaluation of
atorvastatin for the treatment of patients with asthma: a
double-blind randomized clinical trial. Allergy Asthma
Immunol. Res. 4:290–294.
Morimoto, K., W. J. Janssen, M. B. Fessler, K. A. McPhillips,
V. M. Borges, R. P. Bowler, et al. 2006. Lovastatin enhances
clearance of apoptotic cells (efferocytosis) with implications
for chronic obstructive pulmonary disease. J. Immunol.
176:7657–7665.
Murphy, D. M., I. A. Forrest, P. A. Corris, G. E. Johnson,
T. Small, D. Jones, et al. 2008. Simvastatin attenuates release
of neutrophilic and remodeling factors from primary
bronchial epithelial cells derived from stable lung transplant
recipients. Am. J. Physiol. Lung Cell. Mol. Physiol. 294:
592–599.
Noe, B., B. Hagenbuch, B. Stieger, and P. J. Meier. 1997.
Isolation of a multispecific organic anion and cardiac
glycoside transporter from rat brain. Proc. Natl Acad. Sci.
94:10346–10350.
Nozawa, T., K. Imai, J. Nezu, A. Tsuji, and I. Tamai. 2004.
Functional characterization of pH-sensitive organic anion
transporting polypeptide OATP-B in human. J. Pharmacol.
Exp. Ther. 308:438–445.
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
A. A. Zeki et al.
O’Byrne, P. M., and M. D. Inman. 2003. Airway
hyperresponsiveness. Chest 123:411S–416S.
Ohtsuki, S., T. Takizawa, H. Takanaga, S. Hori, K. Hosoya,
and T. Terasaki. 2004. Localization of organic anion
transporting polypeptide 3 (OATP3) in mouse brain
parenchymal and capillary endothelial cells. J. Neurochem.
90:743–749.
Ou, X., B. Wang, F. Wen, Y. Feng, X. Huang, and A. Xao.
2008. Simvastatin attenuates lipopolysaccharide-induced
airway mucus hypersecretion in rats. Chin. Med. J. (Engl.)
121:1680–1687.
Pandya, P., S. Gattani, P. Jain, L. Khirwal, and S. Surana.
2008. Co-solvent evaporation method for enhancement of
solubility and dissolution rate of poorly aqueous soluble
drug simvastatin: in vitro-in vivo evaluation. AAPS
PharmSciTech 9:1247–1252.
Planaguma, A., M. A. Pfeffer, G. Rubin, R. Croze, M. Uddin,
C. N. Serhan, et al. 2010. Lovastatin decreases acute mucosal
inflammation via 15-epi-lipoxin A(4). Mucosal Immunol.
3:270–279.
Prinz, V., U. Laufs, K. Gertz, G. Kronenberg, M. Balkaya, C.
Leithner, et al. 2008. Intravenous rosuvastatin for acute
stroke treatment: an animal study. Stroke 39:433–438.
Sakoda, K., M. Yamamoto, Y. Negishi, J. K. Liao, K. Node,
and Y. Izumi. 2006. Simvastatin decreases IL-6 and IL-8
production in epithelial cells. J. Dent. Res. 85:520–523.
Schaafsma, D., G. Dueck, S. Ghavami, A. Kroeker, M. M.
Mutawe, K. Hauff, et al. 2011. The mevalonate cascade as a
target to suppress extracellular matrix synthesis by human
airway smooth muscle. Am. J. Respir. Cell Mol. Biol.
44:394–403.
Schachter, M. 2005. Chemical, pharmacokinetic and
pharmacodynamic properties of statins: an update. Fundam.
Clin. Pharmacol. 19:117–125.
Seithel, A., S. Eberl, K. Singer, D. Auge, G. Heinkele, N. B.
Wolf, et al. 2007. The influence of macrolide antibiotics on
the uptake of organic anions and drugs mediated by
OATP1B1 and OATP1B3. Drug Metab. Dispos. 35:779–786.
Serajuddin, A. T. M., S. A. Ranadive, and E. M. Mahoney.
1991. Relative lipophilicities, solubilities, and structurepharmacological considerations of 3-hydrory-3methylglutaryl-coenzyme A (HMG-CoA) reductase
lnhibitors pravastatin, lovastatin, mevastatin, and
simvastatin. J. Pharm. Sci. 80:830–833.
Shishehbor, M. H., M.-L. Brennan, R. J. Aviles, X. Fu, M. S.
Penn, D. L. Sprecher, et al. 2003. Statins promote potent
systemic antioxidant effects through specific inflammatory
pathways. Circulation 108:426–431.
Takahashi, S., H. Nakamura, M. Seki, Y. Shiraishi, M.
Yamamoto, M. Furuuchi, et al. 2008. Reversal of elastaseinduced pulmonary emphysema and promotion of alveolar
epithelial cell proliferation by simvastatin in mice.
Am. J. Physiol. Lung Cell. Mol. Physiol. 294:
L882–L890.
Inhaled Statin for the Treatment of Asthma
Takeda, N., M. Kondo, S. Ito, Y. Ito, K. Shimokata, and H.
Kume. 2006. Role of RhoA inactivation in reduced cell
proliferation of human airway smooth muscle by
simvastatin. Am. J. Respir. Cell Mol. Biol. 35:722–729.
Tarhzaoui, K., P. Valensi, G. Leger, F. Cohen-Boulakia, R.
Lestrade, and A. Behar. 2009. Rosuvastatin positively
changes nerve electrophysiology in diabetic rats. Diabetes
Metab. Res. Rev. 25:272–278.
Temelkovski, J., S. P. Hogan, D. P. Shepherd, P. S. Foster, and
R. K. Kumar. 1998. An improved murine model of asthma:
selective airway inflammation, epithelial
lesions and increased methacholine responsiveness following
chronic exposure to aerosolised allergen. Thorax 53:
849–856.
Tse, S. M., L. Li, M. G. Butler, V. Fung, E. O. Kharbanda, E.
K. Larkin, et al. 2013. Statin exposure is associated with
decreased asthma-related emergency department visits and
oral corticosteroid use. Am. J. Respir. Crit. Care Med.
188:1076–1082.
Tse, S. M., S. L. Charland, E. Stanek, V. Herrera, S. Goldfarb,
A. A. Litonjua, et al. 2014. Statin use in asthmatics on
inhaled corticosteroids is associated with decreased risk of
emergency department visits. Curr. Med. Res. Opin. 30:685–
693.
Tulbah, A. S., H. X. Ong, P. Colombo, P. M. Young, and D.
Traini. 2014. Novel simvastatin inhalation formulation and
characterisation. AAPS PharmSciTech 15:956–962.
Turi, G. J., R. Ellis, J. N. Wattie, N. R. Labiris, and M. D.
Inman. 2011. The effects of inhaled house dust mite on
airway barrier function and sensitivity to inhaled
methacholine in mice. Am. J. Physiol. Lung Cell. Mol.
Physiol. 300:L185–L190.
Vaughan, C. J., and A. M. Gotto. 2004. Update on statins:
2003. Circulation 110:886–892.
Vishwanathan, A. 2008. In Vitro Characterization of
Simvastatin Loaded Microspheres in the Polyring Device.
May. 1–79 (Master of Science Thesis. URL: https://
etd.ohiolink.edu/!etd.send_file?
accession=akron1205934178&disposition=inline).
Walters, H. C., A. N. N. L. Craddock, H. Fusegawa, M. C.
Willingham, and P. A. Dawson. 2000. Expression, transport
properties, and chromosomal location of organic anion
transporter subtype 3. Am. J. Physiol. Gastrointest. Liver
Physiol. 279:G1188–G1200.
Wang, C.-Y., P.-Y. Liu, and J. K. Liao. 2008. Pleiotropic effects
of statin therapy: molecular mechanisms and clinical results.
Trends Mol. Med. 14:37–44.
Watts, K. L., and M. A. Spiteri. 2004. Connective tissue
growth factor expression and induction by transforming
growth factor-beta is abrogated by simvastatin via a Rho
signaling mechanism. Am. J. Physiol. Lung Cell. Mol.
Physiol. 287:L1323–L1332.
Watts, K. L., E. M. Sampson, G. S. Schultz, and M. A. Spiteri.
2005. Simvastatin inhibits growth factor expression and
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
2015 | Vol. 3 | Iss. 5 | e12352
Page 21
A. A. Zeki et al.
Inhaled Statin for the Treatment of Asthma
modulates profibrogenic markers in lung fibroblasts. Am. J.
Respir. Cell Mol. Biol. 32:290–300.
Wegesser, T. C., and J. A. Last. 2009. Mouse lung
inflammation after instillation of particulate matter collected
from a working dairy barn. Toxicol. Appl. Pharmacol.
236:348–357.
Xing, X.-Q., S. Duan, X.-W. Wu, Y. Gan, S. P. Zhao, P. Chen,
et al. 2011. Atorvastatin reduces lipopolysaccharide-induced
expression of C-reactive protein in human lung epithelial
cells. Mol. Med. Rep. 4:753–757.
Xu, L., X. W. Dong, L. L. Shen, F. F. Li, J. X. Jiang, R. Cao,
et al. 2012. Simvastatin delivery via inhalation attenuates
airway inflammation in a murine model of asthma. Int.
Immunopharmacol. 12:556–564.
Yeganeh, B., E. Wiechec, S. R. Ande, P. Sharma, A. R.
Moghadam, M. Post, et al. 2014. Targeting the mevalonate
cascade as a new therapeutic approach in heart disease,
cancer and pulmonary disease. Pharmacol. Ther. 143:87–
110.
Yeh, Y. F., and S. L. Huang. 2004. Enhancing effect of
dietary cholesterol and inhibitory effect of pravastatin
on allergic pulmonary inflammation. J. Biomed. Sci. 11:
599–606.
2015 | Vol. 3 | Iss. 5 | e12352
Page 22
Zeki, A. A., L. Franzi, J. Last, and N. J. Kenyon. 2009.
Simvastatin inhibits airway hyperreactivity: implications for
the mevalonate pathway and beyond. Am. J. Respir. Crit.
Care Med. 180:731–740.
Zeki, A. A., J. M. Bratt, M. Rabowsky, J. A. Last, and N. J.
Kenyon. 2010. Simvastatin inhibits goblet cell hyperplasia
and lung arginase in a mouse model of allergic asthma: a
novel treatment for airway remodeling? Transl. Res.
156:335–349.
Zeki, A. A., N. J. Kenyon, and T. Goldkorn. 2011. Statin drugs,
metabolic pathways, and asthma: a therapeutic opportunity
needing further research. Drug Metab. Lett. 5:40–44.
Zeki, A. A., P. Thai, N. J. Kenyon, and R. Wu. 2012.
Differential effects of simvastatin on IL-13-induced cytokine
gene expression in primary mouse tracheal epithelial cells.
Respir. Res. 13:38.
Zeki, A. A., J. Oldham, M. Wilson, O. Fortenko, V. Goyal,
M. Last, et al. 2013. Statin use and asthma control in
patients with severe asthma. BMJ Open 3:1–10.
Zhao, J., B. Maskrey, S. Balzar, K. Chibana, A. Mustovich, H.
Hu, et al. 2009. Interleukin-13 – induced MUC5AC is
regulated by 15-lipoxygenase 1 pathway in human bronchial
epithelial cells. Am. J. Respir. Crit. Care Med. 179:782–790.
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.